This invention relates to NADP linked mixed-function alcohol, aldehyde/ketone oxidoreductases derived from thermophilic anaerobic bacteria and, particularly, to thermostable NADP specific enzymes, a method for producing such enzymes and uses thereof analytically and commercially.
Nicotinamide adenine dinucleotide phosphate (NADP) linked alcohol dehydrogenases have previously been detected in L. mesentroides, C. thermosaccharolyticum and C. kluyeri. Secondary alcohol dehydrogenase activity, often associated with ethanol dehydrogenases, is usually less in these bacteria than is primary alcohol dehydrogenase activity. Such alcohol dehydrogenases have a number of synthetic and diagnostic uses, but commercial preparations lack sufficient heat stability and are too substrate specific for general use.
The NADP specific thermostable alcohol aldehyde, ketone oxidoreductases of the present invention, however, can react at elevated temperatures with a wide range of alcohols, ketones and aldehydes. A unique preference for secondary over primary alcohols is observed according to the following decreasing order of activity: secondary alcohols, ketones and aldehydes, and primary alcohols.
It is an object of this invention to provide for the preparation and use of thermostable NADP linked enzymes from thermally stable microorganisms, such as Thermoanaerobium brockii (T. brockii) and Clostridium thermohydrosulfuricum; and, at least, the partial purification thereof for analytical and commercial use. Other species such as C. thermocellum do not have these enzymes.
The invention will hereinafter be described with respect to the preparation of NADP linked alcohol dehydrogenase or alcohol-aldehyde/ketone oxidoreductase using T. brockii and C. thermohydrosufuricum as representative of the thermostable microorganisms.